Abstract
Immunohistochemical techniques, such as immunofluorescence (IF) staining, enable microscopic imaging of local protein expression within tissue samples. Molecular profiling enabled by IF is critical to understanding pathogenesis and is often involved in complex diagnoses. A recent innovation, known as microscopy with ultraviolet surface excitation (MUSE), uses deep ultraviolet (≈280 nm) illumination to excite labels at the tissue surface, providing equivalent images without fixation, embedding, and sectioning. However, MUSE has not yet been integrated into traditional IF pipelines. This limits its application in more complex diagnoses that rely on protein-specific markers. This paper aims to broaden the applicability of MUSE to multiplex immunohistochemistry using quantum dot nanoparticles. We demonstrate the advantages of quantum dot labels for protein-specific MUSE imaging on both paraffin-embedded and intact tissue, significantly expanding MUSE applicability to protein-specific applications. Furthermore, with recent innovations in three-dimensional ultraviolet fluorescence microscopy, this opens the door to three-dimensional IF imaging with quantum dots using ultraviolet excitation.
Highlights
Chronic diseases such as cancer and neurodegenerative disorders are often accompanied by progressive microscopic alterations in tissue structure and protein composition
While studies of microscopy with ultraviolet surface excitation (MUSE) basics, such as underlying physical phenomena and instrument specifications, have been established [3,21], the reported applications of MUSE have been limited to conventional histological dyes [4,24]
Complex immuno-profiling and automated multiplexing are challenges being addressed to prepare for next-generation precision medicine and digital analysis
Summary
Chronic diseases such as cancer and neurodegenerative disorders are often accompanied by progressive microscopic alterations in tissue structure and protein composition. Assessing these changes is crucial for detecting and monitoring disease [1,2]. In clinical medicine, these changes are often examined through histopathological processing, which integrates multistep tissue preparation, sectioning, chemical labeling, and microscopy to evaluate and grade disease. These changes are often examined through histopathological processing, which integrates multistep tissue preparation, sectioning, chemical labeling, and microscopy to evaluate and grade disease This process is labor-intensive and time-consuming, leading to delays in diagnosis. MUSE can acquire equivalent histological images directly from fresh tissue, enabling integration into conventional histology pipelines while providing faster results at lower cost [4]
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